SUMMARY Precise duplication of the entire human genome requires that many thousands of chromosomal initiation sites, known as DNA replication origins, are utilized exactly once per cell cycle. Origin licensing is accomplished through the DNA loading of precursor helicase complexes, and they are loaded exclusively during the G1 phase of the cell cycle. Licensing too few origins results in incomplete replication, and licensing origins that have already been replicated results in re-replication. Both situations are sources of genome instability that can lead to developmental defects, degeneration, or mutagenesis. Our goal is to understand how the origin licensing process is achieved and how it is organized to ensure appropriate cell proliferation and genome stability. Here, we propose to interrogate licensing control during early differentiation, explore the molecular mechanism of an essential but mysterious licensing factor, and identify the mechanisms and pathways that regulate origin licensing during cell cycle exit. We have designed innovative new tools and assays to quantify and manipulate origin licensing in different human cell lines. We seek answers to questions such as the following: 1) How is licensing coordinated to ensure genome stability over the course of profound G1 changes that occur during differentiation? 2) How does the Cdt1 licensing factor function and how is it controlled? 3) How is licensing blocked during the establishment and maintenance of cell cycle quiescence? Success in answering these questions will shed light on an essential biological process that is fundamental to organismal development and homeostasis. Moreover, we will investigate DNA replication and cell cycle control in cellular settings that have not yet been explored in anticipation of revealing entirely new aspects of cell proliferation control.